Methods for suppressing the endocrine system

ABSTRACT

This invention relates to the parenteral administration of cobalt protoporphyrin and cobalt mesoporphyrin to suppress the endocrine system of animals to achieve physiological effects such as suppression of the production of gonadal or thyroid hormones, control of weight gain and improved protein to fat ratio.

RELATED APPLICATIONS

This application is a continuation in part of copending patentapplication Ser. No. 105,591, filed Nov. 13, 1987 which is acontinuation in part of patent application Ser. No. 927,830, filed Nov.6, 1986 which is, in turn, a continuation in part of patent applicationSer. No. 832,512 filed Feb. 21, 1986, all of which are now abandoned.The latter application is a continuation of patent application Ser. No.708,228 filed Mar. 5, 1985 and now abandoned, which is, in turn acontinuation in part of patent application Ser. No. 363,588 filed Mar.30, 1982, now abandoned.

This invention is concerned with methods of suppressing the endocrinesystem of animals. More particularly, it is concerned with methods forlimiting the activity of the glands of the endocrine system to achievedesirable physiological results such as suppression of hormoneproduction of animals, i.e., living beings including mammals such asman; bovines, particularly beef cattle; sheep; goats; poultry,especially chickens, ducks and turkeys; as well as fish, particularlythose raised in fish farms such as salmon and trout; and, in general,all animals of economic importance in addition to pets by treatment withcobalt protoporphyrin (CoPP) or cobalt mesoporphyrin (CoMP). It isconcerned also with therapeutically useful isotonic compositionscontaining CoPP and/or CoMP. Both of these compounds are known.

The endocrine system is responsible for the production and distributionin animals of hormones which influence such factors as phenotype,homeostasis, size and behavior of the individual as well as weight andweight factors such as protein to fat ratio (P/F). The principal organsof the endocrine system are the hypothalamus, pituitary (anterior andposterior), parathyroid, thyroid, thymus, adrenal, pancreas and gonads(testes in male and ovaries in females). The organs of the systemsecrete one or more hormones which aid in the proper metabolic functionof a target organ. For example, ADH, an antidiuretic hormone, sometimescalled vasopressin secretes from the posterior pituitary gland topromote the reabsorption of water by the kidney. This is thought to beregulated by the hypothalamus.

The hypothalamus is believed to regulate the secretion of theantidiuretic hormone ADH. When specialized cells of the hypothalamussense that the blood lacks sufficient water, ADH is released by thehyopthalamus nerve fibers for secretion by the posterior pituitary. ADHtravels in the bloodstream to its target organ, the kidney. There itpromotes the reabsorption of water so that the blood becomes moredilute. As the blood becomes more dilute because of the reabsorbedwater, this fact is sensed in the hypothalamus, and production of theADH ceases.

In some instances, the target organ of one endocrine gland is a secondgland in the same system. For example, the trophic hormones of theanterior pituitary, e.g., luteinizing hormone (LH), stimulate the gonadsto produce androgens, such as testosterone, estrogen and progesterone.

Growth hormone (GH) produced in the anterior pituitary dramaticallyaffects the phenotype of mammals since it determines the size of theindividual. It causes protein accumulation in almost all of the cells ofthe body. In response to GH, all tissues of the body including bonesgrown. The bones continue to grow during childhood as long as a smallarea of cartilage remains at both ends. Growth ceases when this area ofcartilage is replaced by bone. The long bones have completed theirgrowth at the onset of adulthood.

The amount of GH produced during the early prepubertal years determinesthe size of the individual. If the production is too low, or completelyabsent, the person will become a midget. If too much GH is produced theperson will become a giant.

If the production of GH increases in the adult only the bones of thejaw, eyebrows, nose, fingers and toes can respond. When these begin togrow, the person takes on an unusual appearance characterized by verylarge fingers and toes. This condition is one result ofhyperpituitarism. It is specifically identified as acromegaly. It isbelieved that the biblical figure Goliath had acromegaly. Other suchconditions are known and recognized.

In instances where there is an overproduction of hormones which may bemanifested by undesirable weight gain or increase in growth rate, orwhere there is an undesirable response to normal production of hormones,it would be useful to suppress the production of the offending hormoneor hormones. Such a method has now been discovered.

It has been found that it is possible to suppress the endocrine systemof mammals by treatment with a selected metalloporphyrin thereby makingit possible to treat various human afflictions associated with overactivity of one or more of the glands of the endocrine system. Morespecifically, it has been observed that the administration to animals ofCoPP strongly stimulates heme oxygenase activity while at the same timesuppressing ALA synthetase activity and markedly suppressing theendocrine system.

A particularly valuable aspect of this invention is the ability of CoPPand CoMP to limit the production of the gonadal androgenic andestrogenic hormones such as testosterone and estrogen, and to limit theproduction of the thyroid hormones.

The gonadal hormones have anabolic activity. They and their syntheticanalogs are employed to promote growth. Overproduction of the anabolichormones leads to an increased growth rate associated with increasedfood intake. It has been discovered that one of the immediate effects ofthe administration of CoPP or CoMP to animals is a prompt decrease inappetite and subsequent growth rate. This continues during the periodwhen the concentration of gonadal hormones is below that of untreatedmammals. Surprisingly, the decreased growth rate continues long afterthe gonadal level has returned to normal. In rats, as will be explainedbelow, the low testosterone level continues for about 48 days, but thedecreased growth rate continues for at least 90 to 100 days. In otherrespects the animal appears to be normal except that the P/F ratio isincreased. The period of the decreased testosterone level may becontrolled for selected periods of time by selection of the amount ofCoPP or CoMP administered.

The decreased growth rate observed in animals can be maintained beyondthe 90 days or other originally selected period by furtheradministration of CoPP or CoMP, typically at a lower dosage level. ThusCoPP or CoMP can be used in animals to attain and maintain decreasedgrowth rates, thereby assisting in weight control. As will be explainedmore fully hereinafter, the P/F ratio in the body of an animal treatedwith either of these metallic salts is appreciably increased.

As shown below, CoPP administered to rats results in a very markeddepletion of the thyroid hormones, T₃ and T₄ as well as lower thanexpected levels of the pituitary derived trophic hormone, TSH in animalplasma. Additionally, in such treated animals there is a loss of gonadalhormones such as testosterone and estrogen from plasma and aconcurrently lower than normal level of the pituitary derived trophichormone, LH. It has also been observed on thehypothalamus--pituitary--adrenal axis that there is a suppression ofcortisol in plasma.

These finding indicate that the peripheral endocrine system asrepresented by the thyroid, gonads and adrenals is suppressed and thatthe result is related to suppression of the synthesis or release orpituitary hormones. Microscopic examination of the pituitary indicatethat the thyroid releasing cells (TSH) are markedly smaller in the CoPPtreated animals than in normal control animals.

The figures show the results of studies conducted with rats, and clearlyillustrate the activity of CoPP to suppress the activity of theendocrine system. Similar results are obtained with CoMP.

FIG. 1, panels A and B, depicts graphically the results of experimentsshowing that CoPP suppresses the endocrine system in rats. Specifically,in this example, it is shown that the aspect of the endocrine systeminvolving the pituitary-testicular axis is suppressed. Panel A of FIG. 1shows serum testosterone levels in rats which received 2 doses, eachdoes being 50 um/kg body weight of CoPP with hormone determinationsbeing made 7 days after the initial injection. As can be seen in thecontrol column on the left, serum testosterone levels before treatmentwith CoPP were within the range expected in this animal species. In thegroup of data shown in the right column of this panel (marked CoPP), thelevels of serum testosterone after CoPP administration are all below 0.8ng/ml which is an extraordinary drop in the serum level of this male sexhormone. In panel B on the right, data showing serum LH levels, whichcontrols the synthesis and release of testosterone from the gonads andwhich itself is produced and released by the pituitary underhypothalamic stimulation, are shown. The control group shows the rangeof normal levels of serum LH which are characteristic of this animalspecies. The data for CoPP shown at the right of this panel indicatethat there was no compensatory increase in serum LH release from thepituitary as would normally be expected despite the profound decline inserum testosterone levels shown in the left panel of this figure.

In these studies, the dosage level of CoPP was 50 umol/kg of bodyweight. The number of animals is indicated by the number of dots.

There is known to be a feed-back regulatory endocrine mechanism in whichthe peripheral and central endocrine glands, i.e., pituitary--gonadalaxis in this case, act in concert to keep hormone levels at anappropriate constant level of equilibrium. Thus, if serum testosteronelevels markedly decline, there should be a striking increase in serum LHlevels to restore serum testosterone levels to normal. In contrast tothat expected increase, the CoPP treated animals showed no suchcompensatory increase whatsoever, as can be clearly seen from FIG. 1B.This indicates that the compensatory mechanism for stimulating anenhanced output of serum testosterone had been impaired by the CoPP andthat for reasons which are not clear, the pituitary was unable torelease LH which would be required to compensate the disequilibrium intestosterone production produced by CoPP. Thus the decrease in serumtestosterone levels in these animals was not compensated by a reboundincrease in serum LH which would have brought the serum sex hormonelevels back to normal. This indicates a profound decline in endocrinefunction at both the central and peripheral levels. It strongly suggeststhat the synthetic metalloporphyrin operates at a level even higher thanthe pituitary gland and specifically at some point in the axis betweenthe pituitary-hypothamalus from which releasing hormones (i.e.,LH-releasing hormone etc.) are transmitted to the pituitary where theythen stimulate the release of such trophic hormones as, in this case,LH.

The results with CoMP are similar.

FIG. 2 shows the duration of the suppression of serum testosteronelevels that can be elicited by a single dose of 25 umol/kg body weightof CoPP. As indicated, the bars show the level of serum testosteronewhich on days 14-40 are markedly below normal levels as in consistentwith the data in FIG. 1. Hormone levels gradually revert towards normallevels--but do not enter the normal range, until day 48. Thus a singledose of CoPP is capable of profoundly suppressing the synthesis and therelease of the gonadal sex and growth hormone testosterone and thesynthesis or release of the trophic hormone for gonadal hormoneproduction (LH). This effect can last for 40-48 days after a singleinjection of either of the metalloporphyrins of this invention. Thisability transiently to suppress such an important endocrine complex asthe hyopthalamus-pituitary-gonadal axis is a novel biological actioneffected by administration of the herein disclosed metalloporphyrins andclearly shows the utility of these products for treatment of disordersthat are provoked by excessive levels of the gonadal hormones. In otherstudies the suppression depicted here over a term of 48 days, has beenextended for as long as 9 months by repeated administration of CoPP atperiodic intervals. This clearly indicates that periodic administrationof this synthetic metalloporphyrin will provide a useful means forsuppressing endocrine function for as long as the compound isadministered.

In FIG. 2 the number above each bar is the number of animals tested. Thedotted, horizontal lines represent the range observed for levels oftestosterone in the control animals.

The suppression of the endocrine system resulting in markedly reducedproduction of gonadal and thyroid hormones by CoPP and CoMP isreversible. Once the administration of the selected compound ceases,there is a gradual and complete return to normal of the endocrinesystem. This characteristic of the compounds is of great value inclinical situations in which the degree to which a disorder isresponsive to excessive levels of a particular endocrine secretion isnow known. It can be determined by judicious use--to suppress endocrinefunction--of an appropriate dose of CoPP or CoMP, permitting recovery tonormal when the effect of the compound has receded. However, althoughthe endocrine system by standard measures returns to normal, the growthrate remains at a reduced level for many more weeks.

FIG. 3 shows comparable data for the pituitary-thyroid axis. Panel A ofthe figure shows serum T₄ (thyroxin) levels in animals and the rangeover which they fall normally. The column marked CoPP in this panelshows the markedly diminished levels of serum T₄ which are produced bytreatment of the animals with CoPP (2 times 50 umol/kg b.w.) in the samemanner and with the same dosages indicated for the previous studies withthe sex hormone, testosterone. Panel B shows a similar decrease inlevels of the second major thyroid hormone, T₃, its levels in plasma incontrol animals, and the comparable depleting effect on the plasmalevels of this hormone produced by CoPP. Finally, Panel C shows theserum levels of the trophic hormone for the thyroid gland, i.e., thyroidstimulating hormone (TSH), in both control and CoPP-treated animals. Inanimals including man, when thyroid levels are markedly depleted, asshown in the first two panels of this figure, serum TSH levels willrebound to levels far greater than are seen in control circumstances.However, as panel C indicates, there was of such rebound in theCoPP-treated animals, indicating that the pituitary gland in theseanimals was incapable of responding normally to the decreased plasmalevels of thyroid hormones (T₄ and T₃) that was produced by CoPP.

Similar results are observed with CoMP.

These findings together with those in FIGS. 1 and 2 dealing with thepituitary-gonadal axis clearly indicate a hypothalmic effect of thesynthetic metalloporphyrins of this invention. This effect could bemediated through an action of the compound on the releasing hormones forthe trophic hormones (LH or TSH) which control the peripheral synthesisand secretion of thyroid and gonadal hormones, or by some othermechanism as yet undefined. Thus, in these two major endocrine aces, thesynthetic metalloporphyrins CoPP and CoMP exert a profound suppressiveeffect following administration in relatively small amounts of theselected compound.

It is clear from the results of these studied involving thepituitary-thyroid axis that administration of CoPP is useful to treatmammalian afflictions associated with hyperthyroidism.

FIG. 4 shows the observed weight loss after a single dose of CoPPtreatment of male Sprague-Dawley rats (140-160 g).

The parenteral solutions used in this study and other studies reportedherein can be prepared by dissolving CoPP or CoMP in a small volume of0.1 M NaOH (0.1 ml/ml of the metalloporphyrin solution). The CoPP andCoMP were purchased from Porphyrin Products (Logan, UT.). The pH of thesolution was adjusted to 7.4 with 1 M HCl, and the final volume wasadjusted by the addition of 0.9% NaCl solution. Treatment was bysubcutaneous injection.

In the study, the rats were treated on day zero with the followingsolutions:

    ______________________________________                                               50 umol/kg bw    A                                                            25 umol/kg bw    B                                                             5 umol/kg bw    C                                                            saline           D                                                     ______________________________________                                    

Each point in the figure represents the mean body weight of 6 animals.

It will be seen that after an initial cessation of, or decline in, rateof growth, rats treated with 5-25 umol/kg b.w. gained at a rate parallelto, but below that of saline treated controls. Rats treated with 50umol/kg b.w. lost weight immediately and had not returned to theirstarting weight even after 14 days. However by day 14 they had startedto gain weight at a rate parallel to the controls.

FIG. 5 shows a relationship between testosterone concentration andweight loss. The lower panel, shows the daily weight ±SEM of 6 ratstreated with saline, A, or CoPP at 25 umol/kg b.w. At the timesindicated tail blood was collected and serum testosterone concentrationmeasured (upper panel). The value ±SEM of 6 treated rats B are shown inthe upper panel.

In this study serum testosterone levels declined by about 90% within 4days. Weight gain remained constant during this 4 day period, droppedslightly in the ensuing week, and resumed in succeeding weeks,paralleling controls from about day 20 to day 48. Untreated animalsgained weight steadily throughout the study period. Within the timeperiod from 4 to 25 days, serum testosterone levels remained at markedlylow levels and thereafter returned toward normal which they reachedafter about 40 to 48 days.

Despite the fact that treated animals reverted to a rate of weight gainparalleling that of the controls, even when their serum testosteronelevels had normalized, their total body weights remained consistentlyabout 25% lower than those of untreated animals. It was noted that thisdisparity continued for as long as 90 to 100 days. Similar results areobserved in female rats by studying the relationship between estrogenlevels and weight gain.

Another very significant effect of the administration of CoPP or CoMP isthe marked increase in the P/F ratio in the treated animal.

In one experiment illustrating this unusual and valuable result, 40young Sprague-Dawley rats (160-200 g) were divided into 5 equal groupsof animals. One group was administered saline and served as controlgroup. The remaining 32 animals were treated parenterally with 10umol/kg body weight of CoPP and observed during a 180 day period. Eightof these animals received no further treatment. At the end of two weeksthe remaining 24 animals received a second identical dose of CoPP. Thesequence was repeated at two week intervals utilizing successively 16and 8 animals from the original group. Thus, in addition to the controlgroup, there were four groups of animals who received a total of 10, 20,30 and 40 umol/kg body weight of CoPP respectively. At the end of 180days all animals were sacrificed and the total fat and protein in theirbodies determined. The results are shown in the following table.

                  TABLE 1                                                         ______________________________________                                                         %      %                                                     GROUP            FAT    PROTEIN   P/F RATIO                                   ______________________________________                                        Control                                                                              Saline        19.6   19.6    1.00/1.0                                  1      (CoPP         16.6   20.7    1.25/1.0                                         1 × 10 umol/kg bw)                                               2      (CoPP         14.5   20.4    1.41/1.0                                         2 × 10 umol/kg bw)                                               3      (CoPP         13.3   21.6    1.62/1.0                                         3 × 10 umol/kg bw)                                               4      (CoPP         12.4   21.4    1.73/1.0                                         4 × 10 umol/kg bw)                                               ______________________________________                                    

It will be observed that there has been a constant decrease in thepercent of body fat with increasing doses of CoPP. The ultimate decreasein body weight at the highest dose of CoPP is 41%. This decrease in fatcontent occurs without a statistical increase in the percent proteinresulting in a consequent major increase in the P/F ratio from 1.0/1.0to 1.73/1.0. The same effect can be achieved by one dose of the selectedmetalloporphyrin as with a series of treatments.

Those skilled in the art of animal husbandry will recognize the economicsignificance of this aspect of the invention. It is now possible toproduce economically important animals such as beef cattle and poultrywith edible tissues of less fat content and proportionately increasedprotein content. This is enormous importance in animal husbandry for anumber of reasons. The first of these is that the animals utilize theirfeed more efficiently. Normally it requires more food, and thereforemore expense, to produce a pound of fat than to produce a pound ofprotein. The body of an animal treated in accordance with this inventionwill have less fat and therefore a higher protein to weight ratio thanan untreated animal. The edible tissues of the treated animal will havemore nutritional value than the tissues of untreated animals.

A second important consequence is that the edible meat produced by thetreated animal will have decreased fat content compared to untreatedanimals. Recent studies make it more and more evident that high protein,i.e., lean meat is more healthy for human consumption than meat with ahigh fat content.

It has long been recognized that fish caught in fresh rivers, and lakesor in salt water are more flavorful that the same species raised in fishfarms. This is because the tissues of the latter fish have a higher fatcontent. Application of this invention will, therefore, have a profoundimpact on this industry.

Duck meat is not as popular as chicken or turkey meat. This fact isusually attributed to the high fat content of duck tissue. The use ofCoPP or CoMP should increase the popularity of duck as a comestible.

It appears that the administration of CoPP or CoMP to animals has atleast three effects. These are:

1. A transient decrease in gonadal hormone production with aconcommitant decrease in anabolic or growth promoting activity.

2. A long term appetite suppressing effect possibly by action of thecompounds on some appetite regulatory site in the hypothalmus.

3. An increase in the P/F ration.

It should be noted that endocrine studies in animals have served asvalid and art recognized models of the regulatory mechanisms by whichthe endocrine control systems in humans operate. Thus, for example, theseminal work by Huggins on the role of male sex hormone in thepathogenesis of prostatic hypertrophy and the effects thereon ofcastration (removal of the male sex hormone, testosterone, by removingthe testes) with resulting remission of the hypertrophic prostatecondition was done in dogs. Similarly, the elucidation of the principalregulatory mechanisms for thyroid hormone have been carried out in ratsand other animals, and the results observed have proved to be reliableindicators of analogous results in humans thus permitting accuratepredictions of therapeutic results to be achieved by administration tohumans of agents tested in animals. Thus, these animal models ofendocrine suppression by the CoPP have full relevance to thecircumstances existing in humans and clearly indicate that in selecteddoses the synthetic metalloporphyrins CoPP or CoMP will suppress theendocrine system in humans and other mammals.

The newly discovered ability markedly but transiently to suppress theendocrine system of mammals as described and illustrated herein has veryimportant clinical consequences because there are circumstances in whichthe extend to which a certain condition, for example a hypertrophicgrowth condition in the prostate or thyroid is hormone-sensitive is nowknown. In the past, this problem has often forced the physician toextirpative surgery (castration, thyroidectomy, hypophysectomy, etc.).As a result of this discovery, it is now very simple to administer smalldoses of CoPP or CoMP to suppress the endocrine system (with selectivereplacement of hormones not suspected of being provocative agents) anddetermine the effect of the suppression on the course of the conditionin question. Following the obtaining of such information, therapy isthen continued with the selected metalloporphyrin to maintainsuppression of the endocrine system or selective surgical alteration ofthe appropriate endocrine gland could be carried out if warranted.

The selected compounds of the invention will normally be administeredparenterally, i.e., intraveneously, subcutaneously or intramuscularly insterile, parenteral, isotonic solutions. For such solutions any of awide variety of pharmaceutically acceptable carriers currently in suefor the preparation of parenteral compositions may be employed. Thesolutions may be buffered for example with a phosphate buffer to a pH ofabout 7 to 8, preferably 7.4 to 7.5, and contain solutes such as salineor glucose. The solutions may also contain a polyhydoxy alcohol such asethylene or propylene glycol. The active compounds may also beadministered in solution or suspension in a sterile, inert oil such assesame or safflower oil. A typical dosage regimen will be two separateadministrations per week at a dosage rate of 5 to 50 umol/kg of bodyweight. The physician or veterinarian will determine the specificdosage, and it will depend upon such well understood factors as thecondition under treatment, age, weight and general health of thepatient.

At the levels of administration disclosed and claimed herein all of thedisclosed effects will proceed simultaneously. There will be suppressionof the endocrine system, weight loss and improved P/F ratio. If for anyreason the suppression is not acceptable the necessary counteractinghormones can be separately administered. Alternatively the CoPP or CoMPcan be administered at a lower dosage where endocrine suppression is nolonger a factor as disclosed in copending and commonly owned patentapplication Ser. No. 310,854, filed Feb. 14, 1989.

Typically, an isotonic solution can be prepared by dissolving theselected amount of CoPP or CoMP in 0.1 M aqueous sodium hydroxidesolution, adjusting to the selected pH with 1 M hydrochloric acid, andmaking up to volume with 0.9 aqueous sodium chloride solution. Theconcentration of active agent in the parenteral composition may be fromabout 1 to 25 mg/ml.

The administration of the active agent appears to suppress the completeendocrine system but is particularly valuable for limiting theproduction of gonadal and thyroid hormones, for controlling weight, andfor increasing the P/F ratio in animals. Therefore, as suggested above,if the active agent is administered therapeutically to control oneendocrine function, e.g., secretion of thyroid hormone, there will be aconcurrent treatment with other supplemental hormones, all of which areavailable commercially, to compensate for the suppression of otherendocrine functions.

The suppressive effect of CoPP and CoMP recedes with time and thesubjects revert to their normal state except as to growth rate, ultimatebody weight, and P/F ratio. This occurs during variable periods of timedepending on the dosage of the metalloporphyrin. Thus therapeutictreatment can be continued for selected periods to correct or alleviatea specific condition. It may be interrupted after a first course oftreatment, or it may be continued for differing period of times atdifferent levels.

Inorganic cobalt, iron protoporphyrin (heme), tin protoporphyrin andother metalloporphyrins studied do not have the effect of CoPP and CoMP.It presently appears that the requirements for endocrine systemsuppression are that the metalloporphyrin be capable of stimulating forextended periods of time the catalytic activity of heme oxygenase 5 to15 fold in the liver and perhaps pituitary glands and hypothalmus whilesimultaneously suppressing ALA-synthetase and cytochrome P-450 activityin these organs. These enzymes are the rate limiting enzymes of hemedegradation and heme synthesis respectively. They may be involved in themechanism by which the active agent of the invention suppresses theendocrine system.

What is claimed is:
 1. A method of controlling weight gain in animals inneed of such control which comprises parenteral administration to saidanimal of from 5 to 50 um/kg b.w. of cobalt protoporphyrin or cobaltmesoporphyrin.
 2. A method as in claim 1 wherein cobalt protoporphyrinis administered.
 3. A method as in claim 1 wherein cobalt mesoporphyrinis administered.